Capillary Electrophoresis Mass Spectrometry for Scalable Single-Cell Proteomics

Abstract

Understanding the biochemistry of the cell requires measurement of all the molecules it produces. Single-cell proteomics recently became possible through advances in microanalytical sample preparation, separation by nano-flow liquid chromatography (nanoLC) and capillary electrophoresis (CE), and detection using electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). Here, we demonstrate capillary microsampling CE-ESI-HRMS to be scalable to proteomics across broad cellular dimensions. This study established proof-of-principle using giant, ∼250-µm-diameter cells from embryos of the frog Xenopuslaevis and small, ∼35-µm-diameter neurons in culture from the mouse hippocampus. From ∼18 ng, or ∼0.2% of the total cellular proteome, subcellular analysis of the ventral-animal midline (V11) and equatorial (V12) cells identified 1,133 different proteins in a 16-cell embryo. CE-HRMS achieved ∼20-times higher sensitivity and doubled the speed of instrumental measurements compared to nanoLC, the closest neighboring single-cell technology of choice. Microanalysis was scalable to 722 proteins groups from ∼5 ng of cellular protein digest from identified left dorsal-animal midline cell (D11), supporting sensitivity for smaller cells. Capillary microsampling enabled the isolation and transfer of individual neurons from the culture, identifying 37 proteins between three different cells. A total of 224 proteins were detected from 500 pg of neuronal protein digest, which estimates to a single neuron. Serial dilution returned 157 proteins from sample amounts estimating to about half a cell (250 pg protein) and 70 proteins from ca. a quarter of a neuron (125 pg protein), suggesting sufficient sensitivity for subcellular proteomics. CE-ESI-HRMS complements nanoLC proteomics with scalability, sensitivity, and speed across broad cellular dimensions.

Uncovering biology by single-cell proteomics

“…Capillary electrophoresis coupled with electrospray ionization and high-resolution mass spectrometry (CE-ESI-HRMS) has been demonstrated to have sufficient sensitivity to carry out subcellular proteome analyses 27 . This study analyzed samples that ranged from large, ~250-μm-diameter cells from Xenopus laevis embryos to small, ~35-μm-diameter cultured neurons from mouse hippocampus.…”

Single-Cell Proteomics: The Critical Role of Nanotechnology

“…Using this method also provides less substrate interference and minimal oxidative damage to the cells. However, it is mainly focused on large cell research due to the significant sample loss, unstable interface, and issues of reproducibility [ 29 , 30 ]. In addition, ultrathin-layer gel electrophoresis has also been developed.…”

“…More than 1000 proteins have been detected in isolated HeLa cells using this approach [ 29 ]. However, the suitability of cell lysis, the protein pre-treatment integrity and efficiency, and peptide labeling are major factors affecting the overall number and types of protein identification [ 29 , 30 ]. Multidimensional LC analysis is a sample separation process with at least, and most typically, two different chromatographic separation columns or dimensions.…”

Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics

“…With the rapid development of microfluidic sample preparation and advanced mass spectrometers, single-cell proteomics (scProteomics) has become one of the most active research areas. − Compared with single-cell genomics and transcriptomics, the capability to directly measure single-cell proteomes and their post-translational modifications has great potential to reveal functional phenotypes of cell populations, to understand the cell/embryo development, − to predict disease progression, , and to identify cell type-specific surface markers and therapeutic targets . Two different workflows have been developed and widely used for scProteomics, including label-free ,,− and isobaric-labeling-based quantification methods. ,,− Although the label-free method can provide a more accurate measurement of protein abundance, the analysis throughput is relatively low as every single cell requires a single liquid chromatography-mass spectrometry (LC–MS) run at a time scale of >30 min. , …”